Composition for obtaining biological insecticide comprising strains of bacillus thuringiensis

ABSTRACT

The invention refers to formulae for biologic control of insect plagues in plants, free of chemicals, that include deposited  Bacillus thuringiensis  var. kurstaki strains, as well as crystals from endotoxins derived from said strains. It also refers to the biologic control method for the protection of plants from insect plagues through the application of the said formula on plants, their parts, the soil, or their surroundings.

This application is the National Stage of International ApplicationPCT/CL2012/000060, filed Oct. 16, 2012, which claims priority to Chileanpatent application CL 2542-2011, filed Oct. 13, 2011.

DESCRIPTIVE REPORT

The invention refers to an insecticide composition with a wide actionspectrum on insects that are plague, mainly Lepidoptera, such as Tutaabsoluta, Proeulia spp., Spodoptera frugiperda, Plutella xylostela,Agrotis spp, Lobesia botrana, among other, for which formulae are usedthat include at least one strain selected from the group formed byBacillus thuringiensis var. kurstaki (Bt) strains, NRRL B-50551,deposited by BIO INSUMOS NATIVA LTDA. Parcelo Antilhue Lole 432, Ruta 5Km 2645, Mauk, Chile, with the Agricultural Research Culture Collection(NRRL) International Depositary Authority, 1815 North University Street,Peoria, Ill. 61604 U.S.A., on Aug. 22, 2011, for which the viabilitytest was performed on Aug. 29, 2011 by the International DepositoryAuthority and this deposit is capable of reproduction, deposit numberNRRL B-50552, deposited by BIO INSUMOS NATIVA LTDA. Parcelo AntilhueLole 432, Ruta 5 Km 2645, Mauk, Chile, with the Agricultural ResearchCulture Collection (NRRL) International Depositary Authority, 1815 NorthUniversity Street, Peoria, Ill. 61604 U.S.A., on Aug. 22, 2011, forwhich the viability test was performed on Aug. 29, 2011 by theInternational Depository Authority and this deposit is capable ofreproduction, and deposit number NRRL B-50553, deposited by BIO INSUMOSNATIVA LTDA. Parcelo Antilhue Lole 432, Ruta 5 Km 2645, Mauk, Chile,with the Agricultural Research Culture Collection (NRRL) InternationalDepositary Authority, 1815 North University Street, Peoria, Ill. 61604U.S.A., on Aug. 22, 2011, for which the viability test was performed onAug. 29, 2011 by the International Depository Authority and this depositis capable of reproduction. Hereinafter, these strains shall be calledNRRLB-50551, NRRLB-50552, and NRRL B-50553, respectively.

Many of these plagues have been successfully controlled at world levelby applying different insecticides and by using sexual confusion(Ioratti et al., 2011); however, restrictions in the registration andtolerance of some of the said insecticides which, in general, containchemical products, make it necessary to assess new insecticides ofbiologic origin as alternatives for a comprehensive handling of thisplague.

Micro-biologic insecticide Betk-03 corresponds to a formula containing aBt strain or a mixture of two or three Bt strains for the control ofsome Lepidoptera plague (Vásquez et al., 1995; Niedmann & Meza-Basso,2006), which could affect grape berry moth and be included as analternative for the handling of this plague.

This formula is characterized, in addition to the above mentionedstrains, for including toxins in the form of crystals, as well as viablebacteria spores and other products from bacteria fermentation, jointlyor isolated.

Formulae may be in the form of a capsule to prepare a suspension, abait, a combipack, concentrates, emulsifiables, an encapsulated ormiscible concentrate, or ultra-low volume suspension; ultra-low volumetablets; oil emulsions in water, water in oil for seed treatment;concentrate gel or paste, emulsifiable gel, gel for seed treatment;emulsifiable granules, encapsulated, fine, soluble, and dispersible;macro granules, micro granules; paste; contact, wettable, anddispersible powder for seed treatment; solution for seed treatment,concentrate suspension for seed treatment, without excluding other, andincluding all the formulae above described in any of their conventionalforms. They may be used as carriers, without excluding other argyle,kaolin, talc, zeolite, water, vegetable oils, paraffinic and nonparaffinic minerals, among other agents.

The invention composition allows controlling plagues without the use ofconventional insecticides and, due to its characteristics, it does notpresent environmental restrictions, with the advantage of being used inorganic production or other certification systems, or even appliedimmediately before the harvest.

With regard to the state of the art, we may mention patent ES 2 057 638,which refers to an insecticide composition including:

-   -   (a) Bacillus thuringiensis biomass or only its toxin, and    -   (b) One or more phospholipids or a substance with high        phospholipids content.

The said reference protects a weighted ratio (a) computed as drysubstance in front of (b), included in the range from 1:0.2 to 1:5,containing at least one anionic surfactant selected from fatty acidsesters (C₁₀-C₂₀) with optionally ethoxylated glycerol or sorbitol. Itdescribes a composition where the Bacillus thuringiensis biomassweighted ratio or its toxin regarding the said surfactants is includedin the 10.2 to 1:5 range. It also states that the said Bacillusthuringiensis strains are selected from the donegani, kurstaki, sandiego, and tenebrionis varieties. It also mentions that phospholipidsare selected from phosphatidylcholine, phosphatidylethanolamine,phosphatidylinositol, phosphatidylglycerol and its hydrogenated,hydroxyl and ethoxylated derivatives, indicating that the substance withhigh phospholipids contents is soy lecithin or its derivatives.

The purpose of this invention is to provide a biologic insecticidecomposition for plague control in agricultural, forestry, ornamental,domestic use, and wild plants, especially plagues such as tomato moth(Tuta absoluta), brassica moth (Plutella xylostella), Proeulia (Proeuliaspp), cutworms (Agrotis spp.), alfalfa looper (Spodoptera frugiperda),and grape berry moth (Lobesia botrana) among other plagues.

The composition is formed by at least one strain selected from the groupformed by NRRL B-50551, NRRL B-50552, and NRRL B-50553 strains, as wellas from their possible combinations.

In one form, the composition is formed by at least two strains selectedfrom the group formed by NRRL B-50551, NRRL B-50552, and NRRL B-50553,as well as from their possible combinations.

In an additional form, the composition is formed by a combination ofNRRL B-50551, NRRL B-50552, and NRRL B-50553 strains. In another aspect,the invention also includes toxin crystals produced by NRRL B-50551,NRRL B-50552, and NRRL B-50553 strains.

In the present invention, bacteria insecticide (BI) NRRL B-50551, NRRLB-50552, and NRRL B-50553, their possible combinations, and the formulaethey contain, allow controlling plagues without the use of conventionalinsecticides which, due to their characteristics, do not presentenvironmental restrictions, being possible to use them in conventionalor organic production, or in another certification system.

BRIEF DESCRIPTION OF FIGURES

The following figures provide general information on the resultsdemonstrated and incidence obtained with the use or application onplants cultivated with the use or application of formulae prepared withthe different combinations of bacteria strains NRRL B-50551, NRRLB-50552, and NRRL B-50553; the said figures are an integral part of theinvention.

FIG. 1 shows a diagram with the percentage of bunches damaged byProeulia in orchard handling (reference insecticide) and by B.thuringiensis (BI); bars indicated a standard error. Different lettersshow significant differences between treatments (Tukey p<0.05).

FIG. 2 shows a diagram with the reference chemical insecticide effectand that of BI on the percentage of damage on tomato leaflet attacked byTuta absoluta.

FIG. 3 shows a diagram with the reference chemical insecticide effect,and that of BI on the number of Tuta absoluta live larvae on tomatoplants.

FIG. 4 shows a diagram with the BI application effect on the number ofbunches per tomato plant attacked by Tuta absoluta.

FIG. 5 shows a diagram with the BI application effect on the number offruits, per bunch and total, on tomato plants attacked by Tuta absoluta.

FIG. 6 shows the BI effect on Agrotis spp. larvae mortality in maizeplants.

FIG. 7 shows the BI effect on agrotis larvae mortality compared to achemical and a control insecticide.

DESCRIPTION OF THE INVENTION

This invention refers to an insecticide composition with a wide actionspectrum on insects that are plagues, mainly Lepidoptera, among which wemay find: Tuta absoluta, Proeulia spp., Spodoptera frugiperda, Plutellaxylostela, Agrotis spp, and Lobesia botrana, among other. For thecomposition, formulae were used that include at least one strainselected from the group formed by Bacillus thuringiensis var. kurstakistrains and NRRL B-50551, NRRL B-50552, and NRRL B-50553 strains. Forthis reason, the invention composition is based on the use, withoutdifference, of the said strains, their mixtures, as well as theirtoxins, which may include forms such as a suspension capsule for thetreatment of seeds or a bait in any of its forms, a combi pack in any ofits forms, such as a concentrate, encapsulated, miscible, and ultra-lowvolume; tablets in any of their forms or in the form of ultra-lowvolume, such as oil-in-water or water-in-oil emulsions for the treatmentof seeds in the form of gel or concentrate paste, emulsifiable gel, andgel for the treatment of seeds; in the form or dispersible,encapsulated, fine, soluble, and emulsifiable granules; in the form ofmacro and micro granules; in the form of paste; in the form of contact,wettable, and dispersible powder in the treatment of seeds; in the formof a solution for seed treatment, without excluding other possible formsapplicable in the technical area of this application. The formula canoptionally include carriers for the said micro-organisms, such as clay,kaolin, talcum, zeolite, water, vegetable oils, and paraffinic ornon-paraffinic minerals, without excluding other agents, to carry theinvention Bacillus thuringiensis var. kurstaki strains NRRL B-50551,NRRL B-50552, and NRRL B-50553, which are added to the formula inconcentrations in a range from 10 to 10¹⁰ spores/g; preferably from 10⁵to 10¹⁰ UFC/g.

The invention refers to an insecticide composition with a wide actionspectrum on insects that are plagues, preferably those of theLepidoptera kind, among which we may find: Tuta absoluta, Proeulia spp.,Spodoptera frugiperda, Plutella xylostel, Agrotis spp. Lobesia botrana,among other, for which formulae are used that include at least onestrain selected from the group formed by Bacillus thuringiensis var.kurstaki strains NRRL B-50551 NRRL B-50552, and NRRL B-50553. Thisformula is characterized by the inclusion, in addition to thosementioned above, of a formula that uses toxins in the form of crystal,as well as viable bacteria spores and other products from bacteriafermentation, jointly or individually.

DETAILED DESCRIPTION OF THE INVENTION PREFERRED MODES

Definitions

The following terms and expressions are used along this description, andare known and understood by any expert in the technology.

The term “block”, as used in this description, refers to a cultivationarea with homogeneous management.

The expression “degree days”, as used in this description, refers totemperature accumulation, according to the difference between averagetemperature and threshold temperature for plague development.

The expression “application threshold”, as used in this description,refers to the size of the population requiring handling in order toavoid crop damage.

The expression “real leaves”, as used in this description, refers to theleaf formed by leaflets.

In one mode, the invention refers to a formula for biologic control ofinsect plagues on plants, free of chemicals, where the said formulaincludes at least two strains of Bacillus thuringiensis var. kurstakiselected from the group formed by strains NRRL B-50551, NRRL B-50552,and NRRL B-50553, deposited at ARS-USDA, as well as crystals ofendotoxins derived from the said strains.

In another mode, the invention refers to a formula for biologic controlof insect plagues on plants, free of chemicals, where the said formulaincludes the three strains of Bacillus thuringiensis var. kurstaki, NRRLB-50551, NRRL B-50552, and NRRL B-50553, as well as crystals ofendotoxins derived from the said strains.

In an additional mode, the invention refers to a formula for biologiccontrol of insect plagues on plants, free of chemicals, where the saidformula includes at least one strain of Bacillus thuringiensis var.kurstaki selected from the group formed by strains NRRL B-50551, NRRLB-50552, and NRRL B-50553, as well as crystals of endotoxins derivedfrom the said strains.

In another mode, the invention refers to a formula for biologic controlof insect plagues on plants, free of chemicals, found in a form selectedfrom the group and formed by capsule in suspension for seed treatment; abait in any of its forms; a combi pack in any of its forms,concentrates, emulsifiables; concentrate, encapsulate, miscible, andultra-low volume suspensions; tablets in any of their forms, ofultra-low volume; oil-in-water and water-in-oil emulsions in gel orconcentrate paste form, and emulsifiable gel; dispersible, encapsulated,fine, soluble, and emulsifiable granules; macro or micro granules;paste; contact, wettable, and dispersible powder; a solution, and aconcentrate suspension, among other.

In an additional mode, the invention refers to a formula for biologiccontrol of insect plagues on plants, free of chemicals, where theformula also includes carriers selected from the group formed by clay,kaolin, talcum, zeolite, water, vegetable oils, and paraffinic ornon-paraffinic minerals.

In an additional mode, the invention refers to a formula for biologiccontrol of insect plagues on plants, free of chemicals, which includesconcentrations from 10² UFC/g to 10¹² UFC/g UU of strains NRRL B-50551,NRRL B-50552, and NRRL B-50553 or from their combinations.

In one mode, the invention refers to a formula for biologic control toprotect plants from insect plagues, where the method is the applicationof a formula, according to some of the above-described modes, to plants,their parts, soil, or surroundings.

A preferred mode of the invention considers the application of theformula for biologic control of insect plagues on plants, free ofchemicals, as a biologic control method where the formula is applied onplants and/or their parts, selected from flowers, fruits, seeds, leaves,stems, roots, and rhizomes, among other; protecting plants from pathogeninsect attacks. For the purposes of the invention, it is preferablyunderstood that the said pathogen insects belong to the Lepidopterakind, among which we could mention plague species Tuta absoluta,Proeulia spp., Spodoptera frugiperda, Plutella xylostel, Agrotis spp.,Lobesia botrana.

The following are examples of preferred realization for this invention,which provide information on some of the possible realization forms forthe application of this invention formula in the treatment and controlof some agricultural plagues. These examples represent preferred modes,with no limitation for the rest of possible agricultural applicationsthat an average technical expert could design.

EXAMPLES Example 1

a) Formula with Strain Bacillus thuringiensis var. Kurstaki NRRL B-50551(BI with One Strain)

Preparation of the pesticide formula: a formula was prepared thatincludes bacteria material from strain Bacillus thuringiensis var.kurstaki NRRL B-50551 in the form of dry powder with 10⁸ UFC/gconcentration.

b) Formula with Strain Bacillus thuringiensis var. Kurstaki NRRL B-50552(BI with One Strain)

Preparation of the pesticide formula: a formula was prepared thatincludes bacteria material from strain Bacillus thuringiensis var.kurstaki NRRL B-50552 in the form of dry powder with 10⁸ UFC/gconcentration.

c) Formula with Strain Bacillus thuringiensis var. Kurstaki NRRL B-50553(BI with One Strain)

Preparation of the pesticide formula: a formula was prepared thatincludes bacteria material from strain Bacillus thuringiensis var.kurstaki NRRL B-50553 in the form of dry powder with 10⁸ UFC/gconcentration.

Example 2

In Vitro Assay to Assess the Effect of the Invention Formulae ApplyingThree Individual Strains on the Agrotis spp Plague Mortality.

The assay was carried out by placing Agrotis larvae in their seconddevelopment stage on previously selected tomato leaves. First,treatments were applied with suspensions containing different individualstrains of Bacillus thuringiensis, NRRL B-50551, NRRL B-50552, and NRRLB-50553 at a dose of 1 g/liter, using a formula with 1×10⁸ UFC/gconcentration. After the treatment application, leaves were left to dryin order to place the previously selected larvae, which were kept in agrowth chamber during 144 hours, in a regime of 12-hour light during thenight before being placed on the samples treated. Mortality was assessedvery 24 hours. For each treatment, the assay was repeated 15 times, eachtime using 10 larvae. A comparative assay of the effect was carried outusing a treatment with commercial pesticide Dipel WG (which includesBacillus thuringiensis 6.4% p/p). Data were adjusted according to thecontrol mortality.

Controls: (a group of samples with no treatment whatsoever weresubmitted to the effect of larvae and mortality was assessed).

TABLE 1 Agrotis mortality from the effect of DIPEL, expressed inpercentages and at different times at a dose of 40 ug/ml. hours Strain24 48 72 96 120 144 Control  0.0% e*  0.0% e   5% d   7% d   9% d   12%d Dipel WG (Bacillus  8.3% d 26.7% c 48.3% c 57.8% b 87.7% ab 92.6% athuringiensis 6.4% p/p) Bacillus thuringiensis 90.0% a 90.0% a 90.0% a90.0% a 90.0% a 90.0% a var. kurstaki NRRL B-50551 Bacillusthuringiensis  0.0% e  0.0% e  0.0% e 84.0% ab 87.0% a 90.0% a var.kurstaki NRRL B-50552 Bacillus thuringiensis  0.0% e  0.0% e 20.0% 65.0%b 90.0% a 90.0% a var. kurstaki NRRL B-50553 *Same letters indicate theabsence of Tukey HSD >0.05 significant differences.

According to the results in table 1, it is possible to note that thethree strains, individually, reached mortality levels similar to DIPELafter 144 hours; however, it is also noted that strains under studyreached control levels statistically higher than Dipel after 96 hours,with strain NRRL B-50551 outstanding and reaching mortality levels of90% after 24 hours. This assay shows the superior effect of thisinvention strains compared to the commercial reference, Dipel.

Example 3

Use of BI for Proeulia spp. Control, Compared to the Use of Pesticide(DIPEL®)

This assay was carried out in a location in Totihue, 6th Region, Chile,in a Merlot vineyard, and application was started from the fruit settingto the harvest.

Treatments:

-   -   T1. Orchard management (Dipel WG (Bacillus thuringiensis 6.4%        p/p)): two applications during the season.    -   T2. BI application during blooming, veraison and before leaf        falls, against damage (5 g/l).

Water volume was that normally used in the estate, according to thephenologic condition (around 1000 liters/ha).

Applications were carried out with turbo-nebulizer, where all nozzleswere kept open.

The assay surface corresponded to one hectare per treatment, equal toone block per treatment, separated by a road.

The variety used in the assay was Merlot vines.

Experimental Design

An experimental random design was used, with two treatments and fourrepetitions per treatment, with the application of BI and using theorchard management as control.

Assessments

As a confirmation parameter, fruit damage was assessed (bunches ofgrapes): number of damaged bunches/number of bunches damaged by Proeuliaduring the season.

Assessments were carried out from the fruit setting to harvest, whenfour rows per block were assessed (with treatment and control) of 30plants per row, i.e. one hundred a sixty plants approximately. Eachclearing corresponded to approximately 6 linear meters.

Results

As shown in the diagram in FIG. 1, the percentage of grape bunchesdamaged by Proeulia during treatment with BI was significantly less thanwith application of the commercial reference (T1); this was less thanhalf in the treatment with BI (p<0.05), compared to the control.

These results allow concluding, although 4 applications were carried outand there is still the winter application to be carried out, that BI iseffective in Proeulia control, decreasing damage associated to thisplague in wine grapes when compared to the orchard management(commercial reference).

This way, BI results an effective alternative for the control of thisplague in wine grapes, especially for organic vineyards, considering theoptimistic results of this assay.

Example 4

Assessment of the Insecticide Capacity of Three BI Strains in TomatoCultivation for the Control of Plague Tata absoluta.

This assay assesses the effect of the application of a formula accordingto the invention, which includes the three Bacillus thuringiensisstrains (BI) in order to calculate the level of damage caused by tomatomoth (Tuta absoluta) on tomatoes under organic handling and greenhouseconditions.

Methodology

The assay was carried out in a greenhouse under organic management atPanguilemo Experimental Station belonging to Universidad de Talca (7thRegion, Chile). Tomato plants of the Maria Italia variety were used intheir fourth flower development stage. As control, an equivalentgreenhouse was assessed, to which the formula not containing theinvention Bacillus strains was applied.

The formula used contained the three strains of Bacillus thuringiensisNRRL B-50551, NRRL B-50552, and NRRL B-50553 which, in laboratorycontrolled assays, individually and combined, showed insecticideeffectiveness higher than that of the commercial strain Bacillusthuringiensis var. kurstaki (Dipel WG (Bacillus thuringiensis 6.4% p/p))on tomato moth.

Design and Statistical Analysis:

Completely randomized design with five repetitions of ten plants each.Statistical analysis was carried out with one-way ANDEVA. After findinga significant effect of treatments, a multiple comparison of significantminimum difference was carried out in order to separate means (LSD).Data was submitted to the Kolmogorov-Smirnov and Barlett tests and, inthe cases where assumptions necessary for ANDEVA were not met, they wereadjusted according to the Box-Cox test (damage area and severity).

Treatments

-   -   T0: Control with no applications    -   T1: Comparative application with Dipel WG (Bacillus        thuringiensis 6.4% P/P)    -   T6: BI 10⁸ UFC/g BI application in a dose of 1.5 g/l

Both treatment doses were 1 g/l for their combination.

Treatments were applied once with SOLO® backpack sprayer of 20 litercapacity using an application volume equivalent to 300 l/ha. Treatmentapplication was carried out based on monitoring with pheromone trap(Tuta Stop) and plant damage monitoring; the application threshold was50 individuals/traps/day with 6% of eggs, larvae, or visually observeddamage. In this assay, 50 plants were used, taken at random from theassay plants, after which we waited for the accumulation of degree-days(103.2). This condition occurred in the summer and assessments werecarried out seven days after the application. This allowed the formulaacting on larvae in the first and second stage.

Assessments:

The effect of treatments was assessed by measuring the damaged area(cm²) of leaflets and larva mortality, as well as the number of deadlarvae in 10 real leaves per plant.

Results

TABLE 2 Effect of different tomato moth strains on the damage andmortality. Damaged Mortality area (dead larvaes/ Treatment (cm²/leaflet)10 real leaves/plants) T0: Control  9.167 a* 0.667 a T1 Dipel WG(Bacillus 5.383 a 2.00 b  thuringiensis 6.4% p/p) T2 (BI, mix of 3strains) 14.667 b  1.667 b *Same letter: there is no significantstatistics difference (LSD) P < 0.05.

Table 2 shows the average of each treatment for each parameter assessed.As it may be noted, for the parameter assessed, corresponding to thedamaged area, the best treatment is achieved with the BI inventiveformula, which contains the three strains and results in a less damagedarea compared to the commercial reference Dipel WG (Bacillusthuringiensis 6.4% p/p). With regard to mortality, both treatmentsreached mortality levels higher than those of the control treatment.

Conclusions

Based on the data obtained from this BI invention formula, correspondingto the treatment that includes three strains, it is possible to controlthe Tuta absoluta plague in tomatoes grown in a greenhouse with organicmanagement.

Example 5

In Vitro Tuta absoluta Mortality Assay with BI Insecticide (with One,Two, or Three Strains).

In this assay, Tuta absoluta larvae in their second development stagewere placed on tomato leaves previously treated with a suspensioncontaining different strains of Bacillus thuringiensis (NRRL B-50551,NRRL B-50552, and NRRL B-50553) at a dose of 1 g/liter, using a formulawith a concentration of 1×10⁸ UFC/g. After the treatment, leaves wereleft to dry and larvae were placed on them later; leaves with larvaewere kept in a growth chamber for 144 hours, under a regime of 16 hoursof light and 8 hours of darkness. Larvae mortality was assessed every 24hours. Each treatment included 15 repetitions, each one of them with aset of 10 larvae. Data were adjusted according to the control mortality.

TABLE 3 Agrotis mortality by DIPEL effect (40 mg/ml) expressed inpercentages at different times. hours Strain 24 48 72 96 120 144 Control 0.0% f  0.0% f   0% f   7% e   9% e   12% de Dipel  8.3% e 26.7% d48.3% c 57.8% c 87.7% ab  92.6% a Bt strain NRRL B-50551 80.0% b 80.0% b85.0% ab 90.0% a 90.0% a  90.0% a Bt strain NRRL B-50552  0.0% e  0.0% e 0.0% e 84.0% ab 87.0% ab  90.0% ab Bt strain NRRL B-50553  0.0% e  0.0%e 20.0% 65.0% c 90.0% ab  90.0% ab NRRL B-50551 + NRRL 80.0% b 80.0% b90.0% ab 90.0% a 90.0% ab  90.0% ab B-50552 NRRL B-50552 + NRRL  0.0% e 0.0% e 20.0% 65.0% b 90.0% ab  90.0% a B-50553 NRRL B-50551 + NRRL90.0% ab 93.0% ab 94.0% a 94.0% a 94.0% a  95.0% a B-50553 NRRLB-50551 + NRRL 90.0% ab 95.0% ab 95.0% ab 96.0% a 100.0% a 100.0% aB-50552 + NRRL B-50553

Based on the results in table 3, it is possible to note that the threestrains reach mortality levels similar to the commercial reference used,DIPEL, after 144 hours, for formulae containing individual or combinedstrains, with only two of them or containing the three strains. However,the strains under study reach control levels statistically higher thanDipel after 96 hours, even strain N1 reaches 90% mortality levels after24 hours, showing a higher effect than that of the commercial referencegiven by Dipel. When analyzing the mixtures, it is noted that strain N2and N3 increase their action when combined with N1, which also increasesits actions when combined with the two previous strains.

Example 6

Assessment of BI (with Three Strains) for the Control of Tomato MothCompared to Treatment with a Chemicals Product

Purpose:

This assay assesses the application of a combined formula containing thethree Bacillus thuringiensis strains, NRRL B-50551, NRRL B-50552, andNRRL B-50553, (T2=BI with three strains) in order to calculate the levelof damage caused by the tomato moth under greenhouse conditions incomparison to a commercial product of chemicals origin.

Methodology

The assay was carried out during the 2005-2006 season at a location inQuillota. Applications were carried out on Cv Fortaleza tomato plants,with a first bunch formed and second blooming. Applications were carriedout with a nozzle at 2.5 bar pressure, using an application volumeequivalent to 200 l/ha. Application dates were based on the orchardstandard monitoring protocol; application dates were October 10, 17, and24. Assessments were carried out on October 29 to verify the presence oflive larvae and leaflet damage, while the number of bunches assessmentwas carried out during the harvest (February 2006).

Statistical Design:

The experimental design included three repetitions per treatment and twoaisles per repetition, where 30 plants were assessed in each of them.The statistical analysis was carried out with one-way ANDEVA, with theassessed products as main effect. After finding the treatmentssignificant effect, a multiple comparison of significant minimumdifference was carried out in order to separate averages (LSD). Datawere submitted to Kolmogorov-Smirnov and Barlett tests and, in caseswhere the assumptions necessary for ANDEVA were not met, they wereadjusted according to the Box-Cox test (leaflet damage).

Treatments

-   -   T1: Orchard management (Karate 5 EC, active substance        lambda-cyhalothrin) 25 ml/100 l    -   T2: BI with 3 strains (10⁸ ufc/g) in a dose of 2 g/l

Assessments

Although no differences were noted with regard to the number of damagedleaflets per plant (diagram in FIG. 5), a reduced number of live larvaeper plant was noted (diagram in FIG. 6); this may be because BI does notshow an immediate effect on larvae, allowing them to generate damage inleaflets before they die, for which reason no live larvae were noted onthe plants. However, live larvae were noted among plants under chemicaltreatment, with a contact effect.

Results

In the biologic treatment, a significantly higher performance was notedin the treatment with BI compared to the chemical one, which would bemainly due to the fact that plants with the biologic treatment with notpresent damage in their growth apex, which allowed them a significantlyhigher number of bunches per plant (diagram in FIG. 7).

Conclusions

Based on the data gathered, the BI product shows control over Tutaabsoluta in tomatoes grown in greenhouses under conventional managementwith the dose, wetting volume, and periodicity indicated in this assay.

Example 7

Assessment of BI (Three Strains) for Agrotis spp Control on Maize

Purpose:

Assessing applications of the invention BI formula, which includes thethree Bacillus thuringiensis strains (BI), NRRL B-50551, NRRL B-50552,and NRRL B-50553, in order to calculate the level of damage caused bycutworms on maize (Agrotis spp.) under controlled conditions.

Methodology

Maize was planted in 25×20 cm black plastic bags, in a substrate formedby compost, peat, and perlite in a 1:1:0.5 ratio. Plants were kept inthe laboratory, in day light, in a regime of 12 hours light at 25° C.(with lights on) and a minimum temperature of 16° C. (with lights off).

Larvae (100) were supplied by Universidad de Talca Laboratorio deSanidad Vegetal. In order to perform both tests, it was necessary tocreate a system for the plant to continue developing and the larva befed only from the plant to which it had been inoculated. The followingimage shows the system that met the said requirements.

The system is formed by two 1-liter transparent plastic glasses joinedtogether with tape in an opposite way. The base of the glass in contactwith the soil was cut and the glass was buried in the substrateapproximately 1 cm. A 3×3 cm window was cut in the glass on top in orderto release the plant evapotranspiration. In addition, the base of thisglass was perforated with a hot pin for the same purpose.

This way, we ensured that the larva would feed only from the inoculatedplant. In this assay, the following products were used: Bacillusthuringiensis and Engeo® (Thiametoxam+Lambdacihalotrina) and an absolutecontrol.

TABLE 4 Treatment Product Dose T1 Absolute control — (sterile distilledwater) T2 BI 3 g/L  T5 Engeo ® 1 ml/L

Total design was completely at random, with three repetitions pertreatment (3), with a plant as experimental unit.

Controllers were applied 48 hours before the larvae inoculation to theplant. One larva per plant was applied, and they were assessed every 24hours.

Assessment of Larva Mortality

Clearly, the chemical insecticide has a higher mortality level, reaching100%, while the biologic insecticide reached around 40% mortality, beingstatistically different from the chemical control, as well as fromabsolute control, becoming a viable control alternative, especially insystems where the use of chemical insecticides is restricted orprohibited.

Example 8

Assessment of the BI Concentrations (with Three Strains) on DifferentAgricultural Plagues In Vitro.

In this test, second stage larvae, obtained from eggs of each one of thespecies, were placed on leaves whose surface had been sterilized andthen immersed for 5 minutes in the different treatments for tomato inthe case of Tuta absoluta, grape in the case of Agrotis and Proeulia,bean in the case of Spodoptera, and cabbage in the case of Plutella, allof them placed on previously sterilized 5 cm petri plates. Larvae wereplaced on these leaves inside the plates and kept for 72 hours in aculture chamber at 25° C., with 16 hours day/8 hours night regime; afterthis period, each species mortality level at each dose was assessed.

Treatments consisted in controls with the BI formula with three strains,and in increasing concentrations, from 10 spores/g to 10¹¹ spores per g,with the addition of 1.5 g of each concentration to 1 liter water, whereeach species leaves were immersed.

Each treatment was repeated 10 times, with 10 plates each, with a larvaof each species.

Design was a 12×5 factorial arrangement, where factors were doses andlarva species.

Results

The dose factor, as well as the larva species factor, showed significanteffects. From 10³ concentration, significant differences were noted incontrol without the IB formula with three strains; from 10⁸ UFC/gconcentration no significant mortality increases were noted, being Tutaabsoluta, Agrotis spp and Proeulia larvae the most susceptible ones,while Spodoptea and Plutella showed mortality levels significantly lowerthan the former.

Mortality (%) of different second stage larva species in front ofdifferent bacteria insecticide (BI) formula concentrations under invitro conditions 72 hours after the treatments application. Valuesfollowed by the same letter indicate the absence of significant TukeyHSD differences.

TABLE 5 doses (spores') Insects 0 10 100 10³ 10⁴ 10⁵ 10⁶ 10⁷ 10⁸ 10⁹10¹⁰ 10¹¹ Tuta absoluta 0 h* 0 h 3 h 14 fg 36 de 50 d 64 c 87 b 90 a 92ab 93 a 95 ab Agrotis spp 0 h 0 h 5 h 21 f 41 de 46 d 59 c 83 b 90 ab 90ab 91 ab 94 ab Proeulia spp 0 h 0 h 2 h 27 f 35 e 48 d 74 bc 82 b 94 a95 a 95 a 98 a Spodoptera 0 h 0 h 6 h 12 g 30 e 39 e 71 bc 78 bc 87 b 86b 87 b 87 b frugiperda Plutella 0 h 0 h 0 h 13 g 21 34 e 54 65 c 83 b 84b 84 b 86 b xylosteal *Equal letters indicate the absence of significantTukey Hsd p < 0.05 differences.

Example 9

Effectiveness of BI Formula with Three Strains in the Control of GrapeBerry Moth (Lobesia botrana) Under Laboratory Conditions and atDifferent Concentrations

This assay assessed the effectiveness of the BI invention formula withthree strains, NRRL B-50551, NRRL B-50552, and NRRL B-50553 undercontrolled laboratory conditions, in connection with grape berry moth(Lobesia botrana) control in three concentrations, compared to thestandard micro-biologic insecticide Dipel (B. thuringiensis var.kurstaki strain HD-1).

The assay was carried out at the Universidad de Talca Laboratorio deSanidad Vegetal using Lobesia botrana larvae provided by SAG (ServicioAgricola y Ganadero) and following the quarantine regulations of thesaid institution. As experimental unit, parts of set grape bunches wereused, inside plastic containers covered by a mesh. Pieces of buncheswere selected weighting around 5 g and formed by recently set grapes andtheir corresponding rachis. Wine grape bunches were used (CabernetSauvignon). No insecticide was previously applied to these bunches. Eachrepetition was formed by a group of ten experimental units. Fiverepetitions were carried out for each treatment. In total, 250 larvaewere used for the entire assay.

TABLE 6 Products and doses assessed for the control of grape berry moth(Lobesia botrana) on grape bunches under laboratory conditions. DoseTreatment Products I.A. (g HL⁻¹) 1. CONTROL — — 2. BI (3 strains)Bacillus thuringiensis var. kurstaki 100 3. BI (3 strains) Bacillusthuringiensis var. kurstaki 200 4. BI (3 strains) Bacillus thuringiensisvar. kurstaki 300

Products used in the assay (table 6) were dissolved in distilled waterand applied by immersing the bunches. After draining and drying residuesfrom the product application, a second-third stage larva was placed oneach experimental unity and kept under controlled temperature (20±1° C.)until 85% pupation was completed in the control treatment. During thisperiod, the number of dead larvae was assessed and the presence offeeding signs was recorded (presence of fecal matter) on the 3^(rd),6^(th), 8^(th), and 11^(th) day after application (DDA). Later, adultmortality was assessed to this status, considering that dead pupas thatdid not emerge until 45 DDA. Dead adults were kept at 60° C. for 48hours in order to calculate their dry weight. Finally, a varianceanalysis (Andeva) was carried out of measures repeated in time withlarvae mortality transformed in an angular way. Mortality in adult stateand dry weight were analyzed with Andeva of a factor with adultmortality transformed in an angular way and dry weight withouttransformation. When Andeva found significant differences betweentreatments (P<0.05), averages were separated according to Duncanmultiple comparison test.

Results and Discussion

Treatment with BI formula with 3 strains in its higher dose of 300 gHL⁻¹presented significantly higher mortality that the control treatment fromthe first to the last assessment (table 7). Mortality in these two sametreatments did not present significant differences in any of theassessments (table 7). Treatment with BI formula with 3 strains in itsintermediate dose of 200 gHL⁻¹ presented higher mortality that thecontrol treatment from 3 DDA to 11 DDA, with no significant differenceswith the standard in any of the assessments (table 7). The lowest BIformula with 3 strains assessed showed no significant difference withthe control treatment, and presented less mortality than the standardtreatment in a permanent way (table 7). In the case of survival to adultstatus, doses of 200 and 300 gHL⁻¹ Betk-03 presented mortalitysignificantly higher than those of the control treatment (table 7).

TABLE 7 Grape berry moth (Lobesia botrana) mortality at differentmoments from the application of treatments on grape bunches underlaboratory conditions. Products 3 DDA 6 DDA 8 DDA 11 DDA Adult CONTROL 2.5 a* 2.5 a 5.0 a 7.5 a 17.5 a BI (3 strains) 10.0 ab 17.5 ab 25.0 ab30.0 ab 47.5 b (100 gHL⁻¹) BI (3 strains)  7.5 ab 15.0 ab 37.5 bc 42.5bc  60.0 bc (200 gHL⁻¹) BI (3 strains) 12.5 ab 50.0 b  62.5 bc 65.0 bc72.5 c (300 gHL⁻¹) *Same letters indicate the absence of significantTukey Hsd p < 0.05 differences.

Dry weight or survivor adults from all treatments assessed did not showsignificant differences with the control treatment (table 8).

TABLE 8 Dry weight of grape berry moth (Lobesia botrana) adultssurviving different treatments under laboratory conditions. Adults dryweight Products (g) CONTROL 0.00075 BI (3 strains) (100 gHL⁻¹) 0.00068BI (3 strains) (200 gHL⁻¹) 0.00078 BI (3 strains) (300 gHL⁻¹) 0.00073

Results obtained under laboratory conditions indicate that BImicro-biologic insecticide formula (3 strains) in doses of 200-300 gHL⁻¹presents an effective control of first generation grape berry mothlarvae. Larvae mortality levels reached by the 200-300 gHL⁻¹ dose of BIformula (3 strains) are comparable to those reported in literature onfield assays carried out in different countries in Europe (Ifoulis &Savopoulou-Soultani, 2004, Ruiz de Escudero et al., 2007; Shahini etal., 2010). Also, BI formula (3 strains) in 100-300 gHL⁻¹ doses causes asignificant reduction of adults emergency, although dry weight ofindividuals completing their development are equal to those in thestandard and control treatments. This would indicate that possible sublethal effects would not affect the surviving adults' reproductivecapacity. Under plague low incidence conditions, it is possible to applythe lower dose of 100 gHL⁻¹ of the BI formula (3 strains) in order toreach a significant reduction of emergency in adults in connection withthe control, as under higher infestation conditions it is necessary toapply doses of 200-300 gHL⁻¹ Betk-03.

CONCLUSIONS

1. BI insecticide formula (3 strains) applied in 200-300gHL⁻¹concentrations present grape berry moth larvae (Lobesia botrana)control levels significantly higher than those of the control treatment,with no significant differences with the assessed standard treatment;therefore, it effectively controls this plague on grape under laboratoryconditions, and it is recommended for use in the field under medium andhigh infestation conditions.

2. BI insecticide formula (3 strains) applied in 100 gHL⁻¹concentrations presents adult emergency reduction levels in grape berrymoth (Lobesia botrana) significantly higher than that of the controltreatment; therefore, it effectively controls this plague on grape underlaboratory conditions, and it is recommended for use in the field underlow infestation conditions.

3. No phyto-toxicity symptom on grape bunches treated with the BIinsecticide formula (3 strains) was noted.

The invention claimed is:
 1. A composition for biologic control ofinsect pathogens of plants wherein said composition is free ofconventional insecticides and comprises three Bacillus thuringiensisvariety (var.) kurstaki strains deposited with the Agricultural ResearchService Culture Collection (ARS) with accession numbers NRRL B-50551,NRRL B-50552, and NRRL B-50553 or endotoxins derived from said threestrains.
 2. A composition according to claim 1, wherein the compositioncomprises crystals of endotoxins derived from said strains.
 3. Thecomposition according to claim 1, wherein the composition form isselected from the group consisting of a suspension capsule for seedtreatment; a bait in any of its forms; a combo pack in any of its forms,such as concentrates, emulsifiable, concentrate, encapsulated, ormiscible suspension of ultra-low volume; tablets in any of their forms,of ultralow volume; oil-in-water and water-in-oil emulsions in the formof gel, concentrate paste, or emulsifiable gel; dispersible,encapsulated, fine, soluble, emulsifiable granules; macro granules ormicro granules; paste; contact, wettable or dispersible powder; asolution; and a concentrate suspension.
 4. The composition according toclaim 1, wherein the strains are added to the composition atconcentrations between 10² UFC/g and 10¹² UFC/g UU of each strain. 5.The composition of claim 1, further comprising zeolite as a carrier. 6.A method for protecting a plant from an insect pathogen, the methodcomprising applying a composition according to claim 1 to the plant, apart of the plant, soil adjacent to the plant, or an area surroundingthe plant.
 7. A method according to claim 6, wherein the composition isapplied to a part of the plant selected from a flower, a fruit, a seed,a leaf, a stem, a root, and/or a rhizome.
 8. A method according to claim6, wherein the method protects the plant from a pathogen insect from theorder Lepidoptera.
 9. A method according to claim 8, wherein saidpathogen insect from the order Lepidoptera is selected from the groupconsisting of species Tuta absoluta, Proeulia spp., Spodopterafrugiperda, Plutella xylostella, Agrotis spp., and Lobesia botrana. 10.A method for the protection of a plant from an insect pathogen, themethod comprising applying a composition according to claim 1 to theplant, a part of the plant, soil adjacent to the plant, or an areasurrounding the plant, wherein the composition comprises crystals ofendotoxins derived from said three strains.
 11. A method according toclaim 10, wherein the composition is applied to a part of the plantselected from a flower, a fruit, a seed, a leaf, a stem, a root, and/ora rhizome.
 12. A method according to claim 10, wherein the strains areadded to the composition at concentrations between 10² UFC/g and 10¹²UFC/g UU of each strain.
 13. A method according to claim 10 wherein thecomposition protects the plant from a pathogen insect from the orderLepidoptera.
 14. A method according to claim 13, wherein said pathogeninsect from the order Lepidoptera is selected from the group consistingof species Tuta absoluta, Proeulia spp., Spodoptera frugiperda, Plutellaxylostella, Agrotis spp., and Lobesia botrana.